PROJECT SUMMARY The world has been shocked by the recent COVID-19 pandemic. Macropinocytosis is a form of endocytosis that viruses use to gain entry into cells or to facilitate infection. Our laboratory has the most extensive experience in investigating macropinocytosis in the context of cancer, where it functions as a nutrient acquisition pathway2-12. Macropinocytosis is unique among other endocytic pathways because it is preceded by plasma membrane activity in the form of ruffling. When ruffles fuse with each other they form a macropinosome that encapsulates the surrounding fluid and associated particles. Viruses can use macropinocytosis for cellular internalization or they can hijack macropinocytosis for other aspects of infection. Severe acute respiratory syndrome coronaviruses (SARS-CoVs) induce macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased infection in vitro. These viruses use their Spike protein to signal through the epidermal growth factor receptor (EGFR) to stimulate macropinocytosis, and inhibitors of macropinocytosis or EGFR lead to a reduction in viral spread. While CoVs do not seem to utilize macropinocytosis for entry into the cell, they do use later steps in the macropinosome maturation pathway to augment infection. The maturation of the macropinosome is not very well characterized, but there are some indications that specific signaling pathways are involved. We have extensively studied EGFR-driven macropinocytosis in cancer cells and we can use this expertise to determine strategies for blocking macropinosome maturation and SARS-CoV-2 infection. Our hypothesis is that SARS-CoV-2 uses mature macropinosomes as a way to spread to surrounding cells, either by inducing `macropinosome bursting' as occurs in a process called methuosis, or by traveling extracellularly via recycling macropinosomes. In this proposal, we will explore this hypothesis by 1) taking a candidate approache to blocking macropinosome maturation and 2) determine the mechanism of SARS-CoV-2 viral escape that involves macropinocytosis. This project will be of great significance and impact because, by and large, a detailed picture of what controls macropinosome maturation in the context of EGFR signaling and SARS-CoV-2 is lacking. Moreover, it will constitute the first evaluation of the impact that candidate inhibitors have on macropinosome maturation. Understanding the regulators of macropinosome maturation will shed light on a critical aspect of CoV biology and could lead to new approaches for the treatment of COVID-19.